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The CB2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the mitochondrial intrinsic pathway
Abstract
Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway.
... Notably, while some authors suggested that cannabinoids exert their pro-apoptotic actions via activation of CBRs (including plasma membrane-localized CB1R [85,86], mtCB1R [65] or CB2R [87][88][89]), others have reported alternative pathways acting independently of these receptors [90][91][92][93]. Campbell et al. (2001) showed that apoptosis induced by 5 μM Δ9-THC in cultured cortical neurons was blocked by both the CB1R antagonist AM251 and pertussis toxin, suggesting that Δ9-THC-induced apoptosis is mediated by the G protein subunit Gi/o, following CB1R activation [94]. ...
... Alternatively, some studies have suggested the importance of CB2R in apoptosis. For example, Herrera et al. (2006) showed that the Δ9-THC-induced apoptosis of Jurkat cells was mediated by CB2R [88]. Similarly, Jia et al. (2006) reported that both CB1R and CB2R mediated the cannabinoid-induced apoptosis in the same cell line [85]. ...
... Alternatively, some studies have suggested the importance of CB2R in apoptosis. For example, Herrera et al. (2006) showed that the Δ9-THC-induced apoptosis of Jurkat cells was mediated by CB2R [88]. Similarly, Jia et al. (2006) reported that both CB1R and CB2R mediated the cannabinoid-induced apoptosis in the same cell line [85]. ...
Mitochondria play a critical role in the regulation of several biological processes (e.g., programmed cell death, inflammation, neurotransmission, cell differentiation). In recent years, accumulating findings have evidenced that cannabinoids, a group of endogenous and exogenous (synthetic and plant-derived) psychoactive compounds that bind to cannabinoid receptors, may modulate mitochondrial function and dynamics. As such, mitochondria have gained increasing interest as central mediators in cannabinoids’ pharmacological and toxicological signatures.
Here, we review the mechanisms underlying the cannabinoids’ modulation of mitochondrial activity and dynamics, as well as the potential implications of such mitochondrial processes’ disruption on cell homeostasis and disease. Interestingly, cannabinoids may target different mitochondrial processes (e.g., regulation of intracellular calcium levels, bioenergetic metabolism, apoptosis, and mitochondrial dynamics, including mitochondrial fission and fusion, transport, mitophagy, and biogenesis), by modulating multiple and complex signaling pathways. Of note, the outcome may depend on the experimental models used, as well as the chemical structure, concentration, and exposure settings to the cannabinoid, originating equivocal data. Notably, this interaction seems to represent not only an important feature of cannabinoids’ toxicological signatures, with potential implications for the onset of distinct pathological conditions (e.g., cancer, neurodegenerative diseases, metabolic syndromes), but also an opportunity to develop novel therapeutic strategies for such pathologies, which is also discussed in this review.
... One common pathway activated by phytocannabinoids in different cancer types is the ER-stress pathway, which is one of the main mechanisms to induce apoptosis of glioma, astrocytoma, melanoma, and pancreatic tumor cells [56]. Previous studies on several models of glioma reported that CB1 receptor agonists and, more efficiently, CB2 receptor agonists stimulated the synthesis and accumulation of ceramide, a pro-apoptotic lipid second messenger which leads to the induction of stress protein p8 ( [31,57]; Figure 1). Following this p8 induction, downstream ER-stress-related genes were induced (Figure 1), and as a result, the intrinsic mitochondrial pathway was activated [31,57]. ...
... Previous studies on several models of glioma reported that CB1 receptor agonists and, more efficiently, CB2 receptor agonists stimulated the synthesis and accumulation of ceramide, a pro-apoptotic lipid second messenger which leads to the induction of stress protein p8 ( [31,57]; Figure 1). Following this p8 induction, downstream ER-stress-related genes were induced (Figure 1), and as a result, the intrinsic mitochondrial pathway was activated [31,57]. ...
... Furthermore, treatment with the cannabinoid-receptor synthetic agonist WIN-55,212-2 led to upregulation of the BCL-2 homology 3 (BH3)-only family member BAD, a proapoptotic protein, in response to ceramide activation and the serine/threonine kinase Akt downregulation in glioma cells ([61]; Figure 1). Ceramide is also an important regulator of p38 mitogen-activated protein kinase (MAPK), and previous studies on human leukemia and glioma cells reported that following THC treatment, activation of this pathway induced apoptosis partially via the CB1 and CB2 receptors ( [57,62]; Figure 1). Importantly, in contrast to malignant cells, normal brain cells, such as primary neurons and astrocytes, do not undergo apoptosis or present ceramide accumulation in response to phytocannabinoid treatments [31]. ...
Cancer is a complex family of diseases affecting millions of people worldwide. Gliomas are primary brain tumors that account for ~80% of all malignant brain tumors. Glioblastoma multiforme (GBM) is the most common, invasive, and lethal subtype of glioma. Therapy resistance and intra-GBM tumoral heterogeneity are promoted by subpopulations of glioma stem cells (GSCs). Cannabis sativa produces hundreds of secondary metabolites, such as flavonoids, terpenes, and phytocannabinoids. Around 160 phytocannabinoids have been identified in C. sativa. Cannabis is commonly used to treat various medical conditions, and it is used in the palliative care of cancer patients. The anti-cancer properties of cannabis compounds include cytotoxic, anti-proliferative, and anti-migratory activities on cancer cells and cancer stem cells. The endocannabinoids system is widely distributed in the body, and its dysregulation is associated with different diseases, including various types of cancer. Anti-cancer activities of phytocannabinoids are mediated in glioma cells, at least partially, by the endocannabinoid receptors, triggering various cellular signaling pathways, including the endoplasmic reticulum (ER) stress pathway. Specific combinations of multiple phytocannabinoids act synergistically against cancer cells and may trigger different anti-cancer signaling pathways. Yet, due to scarcity of clinical trials, there remains no solid basis for the anti-cancer therapeutic potential of cannabis compounds.
... Numerous reports have shown that the modulation of CBR activity by cannabinoids inhibits the in vitro and in vivo growth and survival of cancer cells via the production of ceramide, a pro-apoptotic sphingolipid [49,69,[118][119][120]. CBR activation by either ∆ 9 -THC or WIN-55,212-2 drives the intracellular accumulation of ceramide to activate Raf1/ERK signalling, which in turn leads to the production of damaging cellular reactive oxygen species (ROS) (Figures 2 and 3). ...
... CBD and ∆ 9 -THC induced apoptosis in vitro and in vivo using human breast [122] and leukaemic cells [51,120] has been shown to be regulated by ceramide biosynthesis. Ceramide reduces mitochondrial membrane potential by the translocation of BH3-interacting domain death agonist protein, BID, to the mitochondria to release the small hemeprotein, cytochrome C (CYC1) to the cytosol, ultimately activating intrinsic apoptotic pathways via the caspase cascade (CASP8, CASP9, and CASP3) [120,123] ( Figure 2). ...
... CBD and ∆ 9 -THC induced apoptosis in vitro and in vivo using human breast [122] and leukaemic cells [51,120] has been shown to be regulated by ceramide biosynthesis. Ceramide reduces mitochondrial membrane potential by the translocation of BH3-interacting domain death agonist protein, BID, to the mitochondria to release the small hemeprotein, cytochrome C (CYC1) to the cytosol, ultimately activating intrinsic apoptotic pathways via the caspase cascade (CASP8, CASP9, and CASP3) [120,123] ( Figure 2). The overlap between apoptosis and autophagy is potentially regulated by Beclin-1 (BECN1); CBD enhanced the interaction between BECN1 and phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), as this protein-protein interaction cross-regulates the instigation of autophagosomes, and the activation of apoptosis, through direct interactions with anti-apoptosis family members Bcl-2 and/or Bcl-xL (reviewed in [124]). ...
Cannabis has been used to relieve the symptoms of disease for thousands of years. However, social and political biases have limited effective interrogation of the potential benefits of cannabis and polarised public opinion. Further, the medicinal and clinical utility of cannabis is limited by the psychotropic side effects of ∆9-tetrahydrocannabinol (∆9-THC). Evidence is emerging for the therapeutic benefits of cannabis in the treatment of neurological and neurodegenerative diseases, with potential efficacy as an analgesic and antiemetic for the management of cancer-related pain and treatment-related nausea and vomiting, respectively. An increasing number of preclinical studies have established that ∆9-THC can inhibit the growth and proliferation of cancerous cells through the modulation of cannabinoid receptors (CB1R and CB2R), but clinical confirmation remains lacking. In parallel, the anti-cancer properties of non-THC cannabinoids, such as cannabidiol (CBD), are linked to the modulation of non-CB1R/CB2R G-protein-coupled receptors, neurotransmitter receptors, and ligand-regulated transcription factors, which together modulate oncogenic signalling and redox homeostasis. Additional evidence has also demonstrated the anti-inflammatory properties of cannabinoids, and this may prove relevant in the context of peritumoural oedema and the tumour immune microenvironment. This review aims to document the emerging mechanisms of anti-cancer actions of non-THC cannabinoids.
... CB2R is also coupled to Gi/o proteins; thus, the stimulation of CB2R is associated with the inhibition of AC and the cAMP/PKAdependent pathway, as has been observed for CB1R. CB2R stimulation activates MAPK cascades, specifically the ERK and p38 MAPK cascades [24]. Additionally, the activation of CB2R has also been linked to the stimulation of additional intracellular pathways, including the PI3K/Akt pathway [24] ( Figure 5). ...
... CB2R is also coupled to G i / o proteins; thus, the stimulation of CB2R is associated with the inhibition of AC and the cAMP/PKA-dependent pathway, as has been observed for CB1R. CB2R stimulation activates MAPK cascades, specifically the ERK and p38 MAPK cascades [24]. Additionally, the activation of CB2R has also been linked to the stimulation of additional intracellular pathways, including the PI3K/Akt pathway [24] ( Figure 5). ...
... CB2R stimulation activates MAPK cascades, specifically the ERK and p38 MAPK cascades [24]. Additionally, the activation of CB2R has also been linked to the stimulation of additional intracellular pathways, including the PI3K/Akt pathway [24] ( Figure 5). These pathways have been associated with pro-survival effects, as well as with the de novo synthesis of the sphingolipid messenger ceramide [24], which has been linked to the pro-apoptotic effects of CBs. ...
The chief psychoactive constituent of many bioactive phytocannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC) found in hemp, cannabis or marijuana plants are scientifically denoted by the Latin term, Cannabis sativa, acts on cell surface receptors. These receptors are ubiquitously expressed. To date, two cannabinoid receptors have been cloned and characterized. Cannabinoid receptor type 1 (CB1R) is found to serve as the archetype for cannabinoid action in the brain. They have attracted wide interest as the mediator of all psychoactive properties of exogenous and endogenous cannabinoids and they are abundantly expressed on most inhibitory and excitatory neurons. Recent evidence established that cannabinoid receptor type 2 (CB2R) is also expressed in the neurons at both presynaptic and postsynaptic terminals and are involved in neuropsychiatric effects. Distinct types of cells in many regions in the brain express CB2Rs and the cellular origin of CB2Rs that induce specific behavioral effects are emerging. To mimic the bliss effects of marijuana, synthetic cannabinoids (SCBs) have been sprayed onto plant material, and this plant material has been consequently packaged and sold under brand name “Spice” or “K2”. These SCBs have been shown to maintain their affinity and functional activity for CB1R and CB2R and have been shown to cause severe harmful effects when compared to the effects of Δ9-THC. The present review discusses the potential brain mechanisms that are involved in the deleterious effects of SCBs.
... However, CB2-induced reduction to human sperm motility occur in a manner distinct from CB1 [41]. In support of our findings, Herrera et al., (2006) reported that CB2 antagonism abolished pro-apoptotic effects of THC, including hypopolarization of the mitochondrial membrane and cytochrome-c release, in human leukemia cells that only express CB2 [127]. A THC concentration of 1.5μM, which is comparable to the high recreational dose used in the present study, reduced MMP and increased ceramide biosynthesis. ...
... A THC concentration of 1.5μM, which is comparable to the high recreational dose used in the present study, reduced MMP and increased ceramide biosynthesis. Pan-caspase inhibition and selective caspase-8 inhibitors were unable to prevent THC-induced reductions in MMP, suggesting that CB2 receptor activation triggers apoptosis through a ceramide-dependent intrinsic mitochondrial pathway [127]. Treatment of mice sperm with and without CB1 with THC reduced ATP levels and progressive motility at concentrations as low as 1μM [128], suggesting that interactions between THC and sperm mitochondria are not CB1-dependent. ...
Global cannabis use has risen 23% since 2010, with 209 million reported users, most of whom are males of reproductive age. Delta-9-tetrahydrocannabinol (THC), the main psychoactive phytocannabinoid in cannabis, disrupts pro-homeostatic functions of the endocannabinoid system (ECS) within the male reproductive system. The ECS is highly involved in regulating morpho-functional and intrinsic sperm features that are required for fertilization and pre-implantation embryo development. Previous work by our group demonstrated that THC altered sperm capacitation and the transcriptome, including several fertility-associated microRNAs (miRs). Despite the prevalent use of cannabis among males of reproductive age, clinical and pre-clinical research investigating the impact of paternal cannabis on sperm function and the outcomes of artificial reproductive technologies (ARTs) remains inconclusive. Therefore, the present study investigates the impact of in vitro THC exposure on morpho-functional and intrinsic sperm functions, including contributions to embryo development following IVF. Bovine sperm were used as a translational model for human and treated with concentrations of THC that reflect plasma levels after therapeutic (0.032μM), and low (0.32μM)-high (4.8μM) recreational cannabis use. After 6-hours of treatment, THC did not alter the acrosomal reaction, but 4.8μM significantly reduced mitochondrial membrane potential (MMP) (p<0.05), primarily through agonistic interactions with CB-receptors. Fertilization of bovine oocytes with THC-treated sperm did not alter developmental rates, but blastocysts generated from sperm treated with 0.32–4.8μM THC had fewer trophoblasts (p<0.05), while blastocysts generated from sperm exposed to any concentration of THC had fewer cells in the inner cell mass (ICM), particularly within the 0.032μM group (p<0.001). Fertility associated miRs, including miR-346, miR-324, miR-33b, and miR-34c were analyzed in THC-exposed sperm and associated blastocysts generated by IVF, with lower levels of miRs-346, -324, and -33b found in sperm treated with 0.32μM THC, while miR-34c levels were higher in sperm treated with 0.032μM THC (p<0.05). Levels of miR-346 were also lower in sperm treated with 0.032μM THC, but higher in blastocysts generated from sperm exposed to 0.32μM THC (p<0.05). Our findings suggest that THC may alter key morpho-functional and epigenetic sperm factors involved in fertilization and embryo development. This is the first study to demonstrate that sperm exposed to THC in vitro negatively affects embryo quality following IVF.
... This study found that the mRNA and protein expression level of Caspase-3 was significantly increased in the treatment groups with cannabinoids in comparison to the NC group. Consistent with previous studies, cannabinoids upregulated protein Caspase-3, resulting in activated apoptosis 16,32,33,[43][44][45] . For human pancreatic cancer, protein Caspase-3 was activated by cannabinoids in in vitro study, which ultimately resulted in apoptosis 6 . ...
... The Caspase-8 expression has related to the extrinsic apoptotic pathway [27][28][29] . On the other hand, the earlier investigation showed that cannabinoids were involved with the activation of the intrinsic apoptosis pathway 43 . Likewise, the current finding implies that cannabinoids induced apoptosis in the human PDAC xenograft tumors which may not be associated with the activation of Caspase-8. ...
Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.
... Cannabinoids, particularly THC, have been shown to induce apoptosis in various cancer cell lines, including breast, prostate, lung, and colon cancer cells ( Figure 1) [54,55]. The pro-apoptotic effects of cannabinoids are often mediated through the activation of cannabinoid receptors, especially CB1 and CB2, leading to the generation of reactive oxygen species and subsequent cell death signalling cascades [55,58]. Apoptosis is a crucial process for maintaining tissue homeostasis, and its dysregulation can contribute to cancer development and progression. ...
... Cannabinoids, particularly THC, have been shown to induce apoptosis in various cancer cell lines, including breast, prostate, lung, and colon cancer cells ( Figure 1) [54,55]. The pro-apoptotic effects of cannabinoids are often mediated through the activation of cannabinoid receptors, especially CB1 and CB2, leading to the generation of reactive oxygen species and subsequent cell death signalling cascades [55,58]. Cannabinoids have been reported to suppress cancer cell proliferation by arresting the cell cycle and preventing cell division. ...
Emerging research has revealed a complex bidirectional interaction between the gut microbiome and cannabis. Preclinical studies have demonstrated that the gut microbiota can significantly influence the pharmacological effects of cannabinoids. One notable finding is the ability of the gut microbiota to metabolise cannabinoids, including Δ9-tetrahydrocannabinol (THC). This metabolic transformation can alter the potency and duration of cannabinoid effects, potentially impacting their efficacy in cancer treatment. Additionally, the capacity of gut microbiota to activate cannabinoid receptors through the production of secondary bile acids underscores its role in directly influencing the pharmacological activity of cannabinoids. While the literature reveals promising avenues for leveraging the gut microbiome–cannabis axis in cancer therapy, several critical considerations must be accounted for. Firstly, the variability in gut microbiota composition among individuals presents a challenge in developing universal treatment strategies. The diversity in gut microbiota may lead to variations in cannabinoid metabolism and treatment responses, emphasising the need for personalised medicine approaches. The growing interest in understanding how the gut microbiome and cannabis may impact cancer has created a demand for up-to-date, comprehensive reviews to inform researchers and healthcare practitioners. This review provides a timely and invaluable resource by synthesizing the most recent research findings and spotlighting emerging trends. A thorough examination of the literature on the interplay between the gut microbiome and cannabis, specifically focusing on their potential implications for cancer, is presented in this review to devise innovative and effective therapeutic strategies for managing cancer.
... Two studies provided evidence to support subsidiary extrinsic pathway involvement. 37 Alternatively, Olivas-Aguirre et al., demonstrated direct CBD-mitochondrial interaction causing alterations to calcium handling. The group deciphered, through in silico analysis of VDAC-CBD interactions, the specific residues upon which CBD-VDAC interactions were based. ...
... 39 Moreover,Scott et al., demonstrated that this effect could be enhanced through combination with more than one cannabinoid and alongside chemotherapeutic agents.42 Herrera et al., demonstrated that when treating leukaemia cells with THC alongside a selective CBR2 antagonist (SR144528) the pro-apoptotic effect of THC observed when treated in isolation was lost, demonstrating the importance of CBR binding.37 In contrary, Soto-Mercado et al., demonstrated that the cannabinoid, CP55940 can induce apoptosis among Jurkat's independent of CBR binding. ...
Legislative change to cannabis use has generated significant interest into the therapeutic utility of cannabis-derived medical products, particularly in the field of oncology. However, much of this research has focused on adults, leaving physicians and caregivers uncertain as to the safety and efficacy of cannabinoids amongst the pediatric demographic. To this end, the aim of this review is to examine the scope of pharmaceutical cannabis in treatment of pediatric cancer, evaluating its utility as an anti-cancer therapeutic as well as symptom relief agent. This systematic review was conducted following the PRISMA guidelines. 30 included articles comprised of 16 clinical and 14 preclinical studies. There is reasonable evidence to support the use of cannabis in CINV, with plausible utility for other facets of symptomatic relief. Preclinical pediatric cancer models, investigating anti-cancer cannabinoid effect, have provided evidence that may warrant first phase clinical trials.
... The majority of the research regarding pediatric cancers has been conducted in leukemia models, especially in T-cell acute lymphoblastic leukemia (T-ALL), a highly aggressive and chemotherapy-resistant cancer which makes up 15% of all childhood ALL cases. Several groups have shown that cannabinoids induce leukemia cell death both in vitro and in vivo [19][20][21][22][23][24]. Specifically, these studies show that cannabinoids increase intracellular stress and damage mitochondrial membrane potential, resulting in subsequent cytochrome c release and cleavage of caspases 8, 9, 2 and 10 [19,23,24]. ...
... Specifically, these studies show that cannabinoids increase intracellular stress and damage mitochondrial membrane potential, resulting in subsequent cytochrome c release and cleavage of caspases 8, 9, 2 and 10 [19,23,24]. Importantly, ceramide biosynthesis was shown to be essential for cannabinoid-mediated activation of the intrinsic apoptotic pathway [22], as also has been reported in adult glioblastoma models [25]. Additionally, CBD induces ROS production in leukemia cells [26], a common mechanism of action found in other cancers involving an increase in the expression of NAD(P)H oxidases Nox4 and p22 phox [24]. ...
Cannabinoids are a group of chemicals that bind to receptors in the human body and, in turn, modulate the endocannabinoid system (ECS). They can be endogenously produced, synthetic, or derived from the plant Cannabis sativa L. Research over the past several decades has shown that the ECS is a cellular communication network essential to maintain multiple biological functions and the homeostasis of the body. Indeed, cannabinoids have been shown to influence a wide variety of biological effects, including memory, pain, reproduction, bone remodeling or immunity, to name a few. Unsurprisingly, given these broad physiological effects, alterations of the ECS have been found in different diseases, including cancer. In recent years, the medical use of cannabis has been approved in different countries for a variety of human conditions. However, the use of these compounds, specifically as anticancer agents, remains controversial. Studies have shown that cannabinoids do have anticancer activity in different tumor types such as breast cancer, melanoma, lymphoma and adult brain cancer. Specifically, phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) has been shown to induce apoptosis and inhibit proliferation of adult cancer cells, as well as modulate angiogenesis and metastasis. Despite increasing evidence that cannabinoids elicit antitumor effects in adult cancers, there is minimal data available on their effects in children or in pediatric cancers despite public and clinical demand for information. Here we describe a comprehensive and critical review of what is known about the effects of cannabinoids on pediatric cancers, highlight current gaps in knowledge and identify the critical issues that need addressing before considering these promising but controversial drugs for use in pediatric oncology.
... Delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, powerfully modulates immune function in peripheral blood cells 3 , in part, through activating cannabinoid receptor 2 (CBR2) 1,[3][4][5][6] . In vitro studies of cannabis exposure, which contains over 450 compounds, show that it modulates immune function 7-10 , changes cytokine production 8,11,12 , inhibits cell proliferation 2 , and induces apoptosis 13,14 . However, little is known about the mechanisms of in vivo THC exposure on the transcriptomes of distinct types of peripheral blood mononuclear cells (PBMCs) in humans. ...
... (d) Examples of differentially expressed marker genes in each cell cluster. The clusters with similar marker gene profiles in a given cell type were assigned to the same cell type: CD4+ T-cells Clu (2,3,8,13,15,16,19); IL7RCD4+ T-cells Clu(4); CD8+ T-cells Clu (1,7,18,20,21); B cells Clu (6,12,14); NK cells Clu(5) CD14+ monocytes Clu (10,17); FCGR3A monocytes Clu(9) DCs Clu (11). (e) Cell type identification by generalized linear modeling (GLM) cell mapping approach and t-SNE plot. ...
Delta-9-tetrahydrocannabinol (THC) is known to modulate immune response in peripheral blood cells. The mechanisms of THC’s effects on gene expression in human immune cells remains poorly understood. Combining a within-subject design with single cell transcriptome mapping, we report that THC acutely alters gene expression in 15,973 blood cells. We identified 294 transcriptome-wide significant genes among eight cell types including 69 common genes and 225 cell-type-specific genes affected by THC administration, including those genes involving in immune response, cytokine production, cell proliferation and apoptosis. We revealed distinct transcriptomic sub-clusters affected by THC in major immune cell types where THC perturbed cell-type-specific intracellular gene expression correlations. Gene set enrichment analysis further supports the findings of THC’s common and cell-type-specific effects on immune response and cell toxicity. This comprehensive single-cell transcriptomic profiling provides important insights into THC’s acute effects on immune function that may have important medical implications.
... as well as different species and/or cell types, and involves the activation of specific caspases (e.g., caspases 1, 3, 8 and/or 9) ( Zhu et al., 1998;McKallip et al., 2002;Herrera et al., 2006;Jia et al., 2006;Wu et al., 2008;Rieder et al., 2010;Wu et al., 2018). As THC treatment decreased caspase-1 activity within TLR7-activated monocytes, caspase-1-mediated apoptosis is unlikely. ...
... Additional studies are needed to elucidate the mechanism of THC-mediated monocyte apoptosis when stimulated through TLR7, including the contribution of specific caspases. Conversely, the CB2 selective agonist, JWH-015, displayed a minimal effect on monocyte apoptosis, suggesting that THC-mediated monocyte apoptosis at 10µM is likely CB2-independent, which is in contrast with reports identifying THC and CB2 agonists promoting apoptosis through CB2 ( McKallip et al., 2002;Herrera et al., 2006;Jia et al., 2006;Lombard et al., 2007). No apoptotic effects were observed at THC concentrations less than 5µM as well as with JWH-015, suggesting that THC and JWH-015 suppress IL-1b secretion through a non-apoptotic, CB2-dependent mechanism. ...
... 5-epi induces de novo synthesis of Cer through alteration of Serine Palmitoyl-Transferase (SPT) regulation. The mechanism(s) by which CBs induce cell death have been partially delineated and increased de novo synthesis of the sphingolipid, ceramide (Cer), has been identified as a common occurrence [24]. We have previously demonstrated that 5-epi induces de novo synthesis of Cer [12]. ...
Background: Our recent studies have identified a link between sphingolipid metabolites and the induction of a specialized form of regulated cell death termed immunogenic cell death (ICD). We have recently demonstrated that the synthetic cannabinoid (±) 5-epi CP 55,940 (5-epi) stimulates the accumulation of ceramide (Cer), and that inhibition of sphingosine kinase 1 (SphK1) enhances Cer accumulation and ICD-induction in human colorectal cancer (CRC) cell lines. Methods: We employed flow-cytometric, western blot analyses, pharmacological inhibitors of the sphingolipid metabolic pathway and small molecule agonists and antagonists of the CB receptors to further analyze the mechanism by which 5-epi induces Cer accumulation. Results: Herein, and report that 5-epi induces de novo synthesis of Cer primarily through engagement of the cannabinoid receptor 2 (CB2) and depletion of intracellular calcium levels. Moreover, we report that 5-epi stimulates Cer synthesis through dysregulation of the endogenous inhibitor of the de novo Cer pathway, ORMDL3. We also observed a remarkable and specific accumulation of one Cer species, C20:4 Cer, generated predominantly by ceramide synthase 4, as a key factor required for 5-epi-induced ICD. Conclusions: Together, these data indicate that engagement of CB2, by 5-epi, alters regulation of the de novo ceramide synthesis pathway to generate Cer species that mediate ICD.
... However, several studies have reported that CB2R receptor expression is increased in activated T lymphocytes and that its activation decreases their proliferation (74)(75)(76). This is associated with reduced production of proinflammatory cytokines and increased apoptosis (74)(75)(76)(77)(78). In addition, CBR agonists can upregulate immunosuppressive cytokines (79,80) and cause inhibition of chemotaxis (81-83). ...
Triple-negative breast cancer (TNBC) accounts for about 10-20% of all breast cancer cases and is associated with an unfavorable prognosis. Until recently, treatment options for TNBC were limited to chemotherapy. A new successful systemic treatment is immunotherapy with immune checkpoint inhibitors, but new tumor-specific biomarkers are needed to improve patient outcomes. Cannabinoids show antitumor activity in most preclinical studies in TNBC models and do not appear to have adverse effects on chemotherapy. Clinical data are needed to evaluate efficacy and safety in humans. Importantly, the endocannabinoid system is linked to the immune system and immunosuppression. Therefore, cannabinoid receptors could be a potential biomarker for immune checkpoint inhibitor therapy or a novel mechanism to reverse resistance to immunotherapy. In this article, we provide an overview of the currently available information on how cannabinoids may influence standard therapy in TNBC.
... It was shown that the CBN treatment of the human leukaemia cell line Jurkat expressing CB 2 receptors leads to receptordependent ceramide biosynthesis and the downregulation of the Raf-1/mitogen-activated protein kinase/ERK kinase (MEK)/ERK/RSK pathway to induce apoptosis [21]. Similarly, Herrera et al. [85] showed that CBN exerts CB-dependent pro-apoptotic actions in the tumour cells of the human leukaemia cell line Jurka through the ceramide-mediated stimulation of the intrinsic mitochondrial pathway. ER, stress, p8-TRIB3 induction, and autophagy are yet to be fully elucidated in prostate and leukaemia cancer cell lines. ...
Cannabinoids, the bioactive compounds found in Cannabis sativa, have been used for medicinal purposes for centuries, with early discoveries dating back to the BC era (BCE). However, the increased recreational use of cannabis has led to a negative perception of its medicinal and food applications, resulting in legal restrictions in many regions worldwide. Recently, cannabinoids, notably ∆ 9-tetrahydrocannabinol (THC) and cannabidiol (CBD), have gained renewed interest in the medical field due to their anti-cancer properties. These properties include the inhibition of tumour growth and cell invasion, anti-inflammatory effects, and the induction of autophagy and apoptosis. As a result, the use of cannabinoids to treat chemotherapy-associated side effects, like nausea, vomiting, and pain, has increased, and there have been suggestions to implement the large-scale use of cannabinoids in cancer therapy. However, these compounds' cellular and molecular mechanisms of action still need to be fully understood. This review explores the recent evidence of CBD's efficacy as an anti-cancer agent, which is of interest due to its non-psychoactive properties. The current review will also provide an understanding of CBD's common cellular and molecular mechanisms in different cancers. Studies have shown that CBD's anti-cancer activity can be receptor-dependent (CB1, CB2, TRPV, and PPARs) or receptor-independent and can be induced through molecular mechanisms, such as ceramide biosynthesis, the induction of ER stress, and subsequent autophagy and apoptosis. It is projected that these molecular mechanisms will form the basis for the therapeutic applications of CBD. Therefore, it is essential to understand these mechanisms for developing and optimizing pre-clinical CBD-based therapies.
... One class of agents that have been shown to induce cancer cell death through induction of de novo Cer synthesis are the phyto-and synthetic cannabinoids. Indeed, both Δ 9 -THC and cannabidiol (CBD) induce de novo synthesis of Cer (Herrera et al., 2006). Recently, we demonstrated that This article has not been copyedited and formatted. ...
The three arms of the unfolded protein response (UPR) surveil the luminal environment of the endoplasmic reticulum (ER) and transmit information through the lipid bilayer to the cytoplasm to alert the cell of stress conditions within the ER lumen. That same lipid bilayer is the site of de novo synthesis of phospholipids and sphingolipids. Thus, it is no surprise that lipids are modulated by and are modulators of ER stress. Given that sphingolipids have both prosurvival and proapoptotic effects, they also exert opposing effects on life/death decisions in the face of prolonged ER stress detected by the UPR. In this review, we will focus on several recent studies that demonstrate how sphingolipids affect each arm of the UPR. We will also discuss the role of sphingolipids in the process of immunogenic cell death downstream of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiating factor 2α (eIF2α) arm of the UPR. Furthermore, we will discuss strategies to target the sphingolipid metabolic pathway that could potentially act synergistically with agents that induce ER stress as novel anticancer treatments. SIGNIFICANCE STATEMENT: This review provides the readers with a brief discussion of the sphingolipid metabolic pathway and the unfolded protein response. The primary focus of the review is the mechanism(s) by which sphingolipids modulate the endoplasmic reticulum (ER) stress response pathways and the critical role of sphingolipids in the process of immunogenic cell death associated with the ER stress response.
... Cannabinoids, particularly THC, have been shown to induce apoptosis in various cancer cell lines, including breast, prostate, lung, and colon cancer cells ( Figure 2) (Sarfaraz et al., 2008;Velasco et al., 2012). The pro-apoptotic effects of cannabinoids are often mediated through the activation of cannabinoid receptors, especially CB1 and CB2, leading to the generation of reactive oxygen species and subsequent cell death signalling cascades (Herrera et al., 2006;Velasco et al., 2012). ...
Emerging research has revealed a complex bidirectional interaction between the gut microbiome and cannabis. Preclinical studies have demonstrated that the gut microbiota can significantly influence the pharmacological effects of cannabinoids. One notable finding is the ability of the gut microbiota to metabolise cannabinoids, including Δ^9-tetrahydrocannabinol (THC). This metabolic transformation can alter the potency and duration of cannabinoid effects, potentially impacting their efficacy in cancer treatment. Additionally, the capacity of gut microbiota to activate cannabinoid receptors through the production of secondary bile acids underscores its role in directly influencing the pharmacological activity of cannabinoids. While the literature reveals promising avenues for leveraging the gut microbiome-cannabis axis in cancer therapy, several critical considerations must be accounted for. Firstly, the variability in gut microbiota composition among individuals presents a challenge in developing universal treatment strategies. The diversity in gut microbiota may lead to variations in cannabinoid metabolism and treatment responses, emphasising the need for personalised medicine approaches. The growing interest in understanding how the gut microbiome and cannabis may impact cancer has created a demand for up-to-date, comprehensive reviews to inform researchers and healthcare practitioners. This review provides a timely and invaluable resource by synthesizing the most recent research findings and spotlighting emerging trends. A thorough examination of the literature on the interplay between the gut microbiome and cannabis, specifically focusing on their potential implications for cancer, is presented in this review to devise innovative and effective therapeutic strategies for managing cancer.
... The mechanism of protective effects of selective CB 2 agonists at high doses is different from the signaling mentioned above pathways and depends on the activation of PPAR-γ. Several studies indicated that the ceramide level and PPAR-γ activation signaling pathway are responsible for the effects of higher concentrations/doses of CB 2 receptor ligands [16,72,73]. Similarly, it has been demonstrated that the protective effects of high concentrations and amounts of BCP (>5 µM and (>25 mg/kg) were mediated via the CB 2 receptor-PPAR-γ pathway, since the use of PPAR-γ and CB 2 antagonists totally annihilated the protective effects of BCP [14,15,27,32,74]. ...
Multiple Sclerosis (MS) is a prevalent inflammatory disease in which the immune system plays an essential role in the damage, inflammation, and demyelination of central nervous system neurons (CNS). The cannabinoid receptor type 2 (CB2) agonists possess anti-inflammatory effects against noxious stimuli and elevate the neuronal survival rate. We attempted to analyze the protective impact of low doses of β-Caryophyllene (BCP) in experimental autoimmune encephalomyelitis (EAE) mice as a chronic MS model. Immunization of female C57BL/6 mice was achieved through two subcutaneous injections into different areas of the hind flank with an emulsion that consisted of myelin Myelin oligodendrocyte glycoprotein (MOG)35-55 (150 µg) and complete Freund's adjuvant (CFA) (400 µg) with an equal volume. Two intraperitoneal (i.p.) injections of pertussis toxin (300 ng) were performed on the animals on day zero (immunizations day) and 48 h (2nd day) after injection of MOG + CFA. The defensive effect of low doses of BCP (2.5 and 5 mg/kg/d) was investigated in the presence and absence of a CB2 receptor antagonist (1 mg/kg, AM630) in the EAE model. We also examined the pro/anti-inflammatory cytokine levels and the polarization of brain microglia and spleen lymphocytes in EAE animals. According to our findings, low doses of BCP offered protective impacts in the EAE mice treatment in a CB2 receptor-dependent way. In addition, according to results, BCP decreased the pathological and clinical defects in EAE mice via modulating adaptive (lymphocytes) and innate (microglia) immune systems from inflammatory phenotypes (M1/Th1/Th17) to anti-inflammatory (M2/Th2/Treg) phenotypes. Additionally, BCP elevated the anti-inflammatory cytokine IL-10 and reduced blood inflammatory cytokines. BCP almost targeted the systemic immune system more than the CNS immune system. Thus, a low dose of BCP can be suggested as a therapeutic effect on MS treatment with potent anti-inflammatory effects and possibly lower toxicity.
... In endothelial cells, CB 2 activation leads to phospholipase C-mediated Ca 2+ release from the endoplasmic reticulum [48]. They are also involved in apoptotic modulation, where ceramide-dependent activation of the mitochondrial intrinsic pathway ultimately results in tumor cell apoptosis [49]. Interestingly, CB 2 activation promotes the expression of anti-apoptotic and neuroprotective p-CREB via the activation of ERK1/2 [50] and AMPK [51], and inhibits pro-apoptotic GSK3β through the PI3K/Akt pathway [52]. ...
Sleep is an essential biological phase of our daily life cycle and is necessary for maintaining homeostasis, alertness, metabolism, cognition, and other key functions across the animal kingdom. Dysfunctional sleep leads to deleterious effects on health, mood, and cognition, including memory deficits and an increased risk of diabetes, stroke, and neurological disorders. Sleep is regulated by several brain neuronal circuits, neuromodulators, and neurotransmitters, where cannabinoids have been increasingly found to play a part in its modulation. Cannabinoids, a group of lipid metabolites, are regulatory molecules that bind mainly to cannabinoid receptors (CB1 and CB2). Much evidence supports the role of cannabinoid receptors in the modulation of sleep, where their alteration exhibits sleep-promoting effects, including an increase in non-rapid-eye movement sleep and a reduction in sleep latency. However, the pharmacological alteration of CB1 receptors is associated with adverse psychotropic effects, which are not exhibited in CB2 receptor alteration. Hence, selective alteration of CB2 receptors is also of clinical importance, where it could potentially be used in treating sleep disorders. Thus, it is crucial to understand the neurobiological basis of cannabinoids in sleep physiology. In this review article, the alteration of the endocannabinoid system by various cannabinoids and their respective effects on the sleep-wake cycle are discussed based on recent findings. The mechanisms of the cannabinoid receptors on sleep and wakefulness are also explored for their clinical implications and potential therapeutic use on sleep disorders.
... Em relação aos estudos realizados in vivo 14 O uso da Cannabis sativa vem cada vez mais ganhando espaço na área terapêutica, sendo que já existem diversos estudos acerca do potencial terapêutico desta planta para o tratamento de diferentes doenças, incluindo leucemias, que são patologias semelhantes aos linfomas 7,16 . Todos os estudos abordados sugeriram que os fitocanabinoides derivados da Cannabis sativa possuem efetividade no tratamento de linfoma não Hodgkin, porém, os mecanismos desta atividade antitumoral ainda não são bem conhecidos. ...
Introdução: A Cannabis sativa é uma planta de grande interesse medicinal devido seus diversos constituintes com propriedades farmacológicas, sendo os principais o canabidiol (CBD) e o Δ9-tetrahidrocanabidiol (Δ9-THC). Uma série de estudos relatam o potencial dos fitocanabinoides para diversas doenças, incluindo o câncer, sendo os linfomas cânceres que afetam, principalmente, as células de defesa do organismo. Objetivos: Analisar o que há de evidências científicas a respeito do potencial antitumoral dos fitocanabinoides contra os linfomas. Materiais e Métodos: Trata-se de uma revisão integrativa da literatura, utilizando artigos indexados nas bases de dados: PubMed, BVS, SciELO, Periódicos CAPES e Science Direct, através dos descritores em inglês: “Cannabis”, “cannabidiol”, “lymphoma” e “apoptosis”, e português: “Cannabis”, “canabidiol”, “linfoma” e “apoptose”, combinados aos operadores booleanos AND e OR, publicados até agosto de 2021. Resultados: Foram encontrados seis estudos, sendo estes, realizados in vivo, in vitro, ex vivo, ou ensaio clínico. O fitocanabinoide mais estudado foi o CBD e todos os estudos selecionados analisaram apenas linfomas não Hodgkin. Todos os ensaios demonstraram a efetividade do uso dos fitocanabinoides no tratamento de linfomas não Hodgkin, sendo o efeito antitumoral demonstrado através da indução de apoptose, citotoxicidade, antiproliferação, e no ensaio clínico por redirecionamento das células malignas para órgãos secundários. Conclusões: A maioria dos resultados obtidos são provindos de pesquisas in vitro ou com animais, sendo detectada uma carência de estudos clínicos, além disso, são necessários mais estudos em relação aos mecanismos envolvidos na ação dos fitocanabinoides contra linfomas e outros cânceres, visto que ainda são pouco conhecidos.
... Another study also found that THC can induce apoptosis of Jurkat cells via ceramide biosynthesis, triggering the same intrinsic pathway via the mitochondria. Jurkat cells, as immune cells, express primarily CB2 receptors, and all of the above effects were found to be mediated via CB2 [98]. This is notable, as CB1 is the receptor through which THC-induced psychoactivity, a major concern when considering the use of cannabis in pediatric populations, is mediated. ...
The antineoplastic effects of cannabis have been known since 1975. Since the identification of the components of the endogenous cannabinoid system (ECS) in the 1990s, research into the potential of cannabinoids as medicine has exploded, including in anti-cancer research. However, nearly all of this research has been on adults. Physicians and governing bodies remain cautious in recommending the use of cannabis in children, since the ECS develops early in life and data about cannabis exposure in utero show negative outcomes. However, there exist many published cases of use of cannabis in children to treat pediatric epilepsy and chemotherapy-induced nausea and vomiting (CINV) that show both the safety and efficacy of cannabis in pediatric populations. Additionally, promising preclinical evidence showing that cannabis has anti-cancer effects on pediatric cancer warrants further investigation of cannabis’ use in pediatric cancer patients, as well as other populations of pediatric patients. This review aims to examine the evidence regarding the potential clinical utility of cannabis as an anti-cancer treatment in children by summarizing what is currently known about uses of medical cannabis in children, particularly regarding its anti-cancer potential.
... Although CB 2 receptor expression was barely detected in circulating T lymphocytes (Figure 1), several studies reported that CB 2 receptor expression is increased in activated T lymphocytes and that its activation decreases their proliferation (Borner et al., 2009;Cencioni et al., 2010;Capozzi et al., 2018). This is accompanied with decreased IL-2 production and increased apoptosis (Herrera et al., 2006;Borner et al., 2009;Cencioni et al., 2010;Capozzi et al., 2018;Huang et al., 2019). Interestingly, CB 2 receptor activation seems to exert divergent effects depending on the T lymphocyte subtype with the tendency to decrease human Th1 and Th17 functions, while promoting those of Th2. ...
The cannabinoid CB2 receptor was cloned from the promyeloid cell line HL-60 and is notably expressed in most, if not all leukocyte types. This relatively restricted localization, combined to the absence of psychotropic effects following its activation, make it an attractive drug target for inflammatory and autoimmune diseases. Therefore, there has been an increasing interest in the past decades to identify precisely which immune cells express the CB2 receptor and what are the consequences of such activation. Herein, we provide new data on the expression of both CB1 and CB2 receptors by human blood leukocytes and discuss the impact of CB2 receptor activation in human leukocytes. While the expression of the CB2 mRNA can be detected in eosinophils, neutrophils, monocytes, B and T lymphocytes, this receptor is most abundant in human eosinophils and B lymphocytes. We also review the evidence obtained from primary human leukocytes and immortalized cell lines regarding the regulation of their functions by the CB2 receptor, which underscore the urgent need to deepen our understanding of the CB2 receptor as an immunoregulator in humans.
... The effects of cannabinoids are mainly mediated via CB1 and CB2 activation. While both isoforms are ubiquitously expressed throughout the body, CB1 is found predominantly in the central nervous system [82], while CB2 is found in the periphery within immune cells such as B lymphocytes and macrophages [83][84][85]. Both CB1 and CB2 are G proteincoupled receptors that modulate several signaling pathways. ...
Cannabis use during pregnancy has continued to rise, particularly in developed countries, as a result of the trend towards legalization and lack of consistent, evidence-based knowledge on the matter. While there is conflicting data regarding whether cannabis use during pregnancy leads to adverse outcomes such as stillbirth, preterm birth, low birthweight, or increased admission to neonatal intensive care units, investigations into long-term effects on the offspring’s health are limited. Historically, studies have focused on the neurobehavioral effects of prenatal cannabis exposure on the offspring. The effects of cannabis on other physiological aspects of the developing fetus have received less attention. Importantly, our knowledge about cannabinoid signaling in the placenta is also limited. The endocannabinoid system (ECS) is present at early stages of development and represents a potential target for exogenous cannabinoids in utero. The ECS is expressed in a broad range of tissues and influences a spectrum of cellular functions. The aim of this review is to explore the current evidence surrounding the effects of prenatal exposure to cannabinoids and the role of the ECS in the placenta and the developing fetus.
... CBD administration also determined alterations on cell morphology, ER, and Golgi, thus reducing cell size and inducing vacuolation [153]. Therefore, CBs may increase intracellular stress and modify mitochondrial membrane potential, causing cytochrome c discharge and cleavage of caspases 8, 9, 2, and 10, up to cell death [150][151][152][153][157][158][159][160][161][162][163]. ...
The endocannabinoid system (ECS) is a composite cell-signaling system that allows endogenous cannabinoid ligands to control cell functions through the interaction with cannabinoid receptors. Modifications of the ECS might contribute to the pathogenesis of different diseases, including cancers. However, the use of these compounds as antitumor agents remains debatable. Pre-clinical experimental studies have shown that cannabinoids (CBs) might be effective for the treatment of hematological malignancies, such as leukemia and lymphoma. Specifically, CBs may activate programmed cell death mechanisms, thus blocking cancer cell growth, and may modulate both autophagy and angiogenesis. Therefore, CBs may have significant anti-tumor effects in hematologic diseases and may synergistically act with chemotherapeutic agents, possibly also reducing chemoresistance. Moreover, targeting ECS might be considered as a novel approach for the management of graft versus host disease, thus reducing some symptoms such as anorexia, cachexia, fatigue, anxiety, depression, and neuropathic pain. The aim of the present review is to collect the state of the art of CBs effects on hematological tumors, thus focusing on the essential topics that might be useful before moving into the clinical practice.
... Our current findings demonstrate that synthetic cannabinoids induce apoptosis in a panel of human glioblastoma cells by promoting the collapse of mitochondrial potential. The contribution of the mitochondrial pathway to apoptosis triggered by a selective CB2 receptor agonist has been demonstrated in Jurkat cells [52], but our report is the first to show this mechanism in non-immune system cells. In tested glioblastoma cell lines expressing CB1 and CB2 cannabinoid receptors, both WIN55,212-2 and JWH133 disturbed the mitochondrial membrane potential and induced a subsequent cleavage of caspase 9 leading to the execution of apoptotic cell death using the same mitochondria-dependent pathway. ...
Glioblastomas (GBMs) are aggressive brain tumors with frequent genetic alterations in TP53 and PTEN tumor suppressor genes rendering resistance to standard chemotherapeutics. Cannabinoid type 1 and 2 (CB1/CB2) receptor expression in GBMs and antitumor activity of cannabinoids in glioma cells and animal models, raised promises for a targeted treatment of these tumors. The susceptibility of human glioma cells to CB2-agonists and their mechanism of action are not fully elucidated. We determined CB1 and CB2 expression in 14 low-grade and 21 high-grade tumor biopsies, GBM-derived primary cultures and established cell lines. The non-selective CB receptor agonist WIN55,212-2 (but not its inactive enantiomer) or the CB2-selective agonist JWH133 induced apoptosis in patient-derived glioma cultures and five established glioma cell lines despite p53 and/or PTEN deficiency. Growth inhibitory efficacy of cannabinoids correlated with CB1/CB2 expression (EC50 WIN55,212-2: 7.36–15.70 µM, JWH133: 12.15–143.20 µM). Treatment with WIN55,212-2 or JWH133 led to activation of the apoptotic mitochondrial pathway and DNA fragmentation. Synthetic cannabinoid action was associated with the induction of autophagy and knockdown of autophagy genes augmented cannabinoid-induced apoptotic cell death. The high susceptibility of human glioblastoma cells to synthetic cannabinoids, despite genetic defects contributing to apoptosis resistance, makes cannabinoids promising anti-glioma therapeutics.
... Por último, resumimos algunos mecanismos moleculares por los cuales la terapia con cannabinoides demuestra distintos efectos antitumorales [20][21][22] (tabla 4). ...
The objective of this review is to check the possibility and plausibility of using selective
cannabinoid agonists/antagonists as antitumor drugs, because the neoplastic cells have
demonstrated an altered expression of these ligands. Based on the study of the endocannabinoid system and its relevance as a superior homeostatic modulator in different brain functions or cognitive ones, various lines of research started studying the mechanisms of action and cellular response of the cannabinoid receptors type 1 and type 2. In this respect it has been shown that the cannabinoid receptor agonist drugs (cannabimemetic) are capable of inhibiting mitosis of cancer cells. Multiple studies in various cancer cells, where a xenotransplant is done in rats with tumor from human cells, they show a decrease in cell growth, dose-dependent effect, thus as on the viability of cancer cells. Is completely encouraging to study more thoroughly this new therapeutic approach against cancer, the impact that might have to deploy synergistic therapies based on cannabinoids and chemotherapy against the most prevalent malignancies in the world, could offer a more complete treatment. This review checks the neoplasm that are sensitive to be treated with cannabinoids, the endocannabinoide system in cancer, the adverse effects of
the cannabinoids in cancer, and the most relevant studies that justify this new therapeutic approach against cancer.
... It has also been shown that THC increases the accumulation of ceramides in cells, while inhibition of their synthesis abolishes the proapoptotic effect of THC. This implies that the accumulation of ceramides under the influence of THC is the reason for the proapoptotic action of THC [142]. An increase in ceramide production also induces ER stress and apoptosis, resulting from the activation of CB1/CB2 receptors by THC [143]. ...
Apoptosis is the physiological mechanism of cell death and can be modulated by endogenous and exogenous factors, including stress and metabolic alterations. Reactive oxygen species (ROS), as well as ROS-dependent lipid peroxidation products (including isoprostanes and reactive aldehydes including 4-hydroxynonenal) are proapoptotic factors. These mediators can activate apoptosis via mitochondrial-, receptor-, or ER stress-dependent pathways. Phospholipid metabolism is also an essential regulator of apoptosis, producing the proapoptotic prostaglandins of the PGD and PGJ series, as well as the antiapoptotic prostaglandins of the PGE series, but also 12-HETE and 20-HETE. The effect of endocannabinoids and phytocannabinoids on apoptosis depends on cell type-specific differences. Cells where cannabinoid receptor type 1 (CB1) is the dominant cannabinoid receptor, as well as cells with high cyclooxygenase (COX) activity, undergo apoptosis after the administration of cannabinoids. In contrast, in cells where CB2 receptors dominate, and cells with low COX activity, cannabinoids act in a cytoprotective manner. Therefore, cell type-specific differences in the pro- and antiapoptotic effects of lipids and their (oxidative) products might reveal new options for differential bioanalysis between normal, functional, and degenerating or malignant cells, and better integrative biomedical treatments of major stress-associated diseases.
... Between these proteins and transcription factors, there is a series of feedback circuits. TRB3 subsequently inhibits the PI3K/AKT/mTOR signal pathway at a level between mTORC2 and ACT (57,58,60,61,62,63,64,65,66,67). The signaling pathway p8/ATF4/CHOP/TRB3, followed by the inhibition of the PI3K/AKT/mTOR cascade, is probably the most important antitumoral mechanism of cannabinoids (45,57,58). ...
Aim:
The aim of this review article is to summarize current knowledge about the role of cannabinoids and cannabinoid receptors in tumor disease modulation and to evaluate comprehensively the use of cannabinoids in cancer patients.
Method:
According to the PRISMA protocol, we have included data from a total of 105 articles.
Results:
Cannabinoids affect cancer progression by three mechanisms. The most important mechanism is the stimulation of autophagy and affecting the signaling pathways leading to apoptosis. The most important mechanism of this process is the accumulation of ceramide. Cannabinoids also stimulate apoptosis by mechanisms independent of autophagy. Other mechanisms by which cannabinoids affect tumor growth are inhibition of tumor angiogenesis, invasiveness, metastasis, and the modulation of the anti-tumor immune response.
Conclusion:
In addition to the symptomatic therapy of cancer patients, the antitumor effects of cannabinoids (whether in monotherapy or in combination with other cancer therapies) have promising potential in the treatment of cancer patients. More clinical trials are needed to demonstrate the antitumor effect of cannabinoids (Tab. 1, Fig. 1, Ref. 167).
... Administration of Can caused an upregulation of caspase-3 and caspase-9 mRNA expression and cyt c levels in the hippocampus and PFC of rats. These findings corroborate a previous study reporting that THC induced apoptosis via stimulation of the mitochondrial intrinsic pathway (Herrera et al., 2006). However, Nan increased caspase-3 and caspase-8 mRNA expression in the hippocampus of rats. ...
... CB2-selective agonists regulate cell proliferation, differentiation, transformation, and death by stimulating major components of the MAPK pathway, including ERK1/2, p38, and c-Jun N-terminal kinases (40). CB2 also induces apoptosis, necrosis, and autophagy through modulations of the Akt-phosphoinositide 3-kinase pathways, as well as modulating arrestin activity and ceramide production (41)(42)(43)(44). Structurally, CB2 shares approximately 50% amino acid homology with CB1 in the transmembrane region; however, these two receptors are functionally distinct, as they exhibit differences in binding affinity for cannabinoid agonists and antagonists (45). ...
This paper reviews the endocannabinoid system and focuses on the role of endocannabinoids in bone metabolism and their potential use in the management of conditions associated with bone loss.
Context:
The endocannabinoid system uses tissue-specific lipid ligands and G protein-coupled transmembrane receptors to regulate neurological, metabolic, and immune responses. Recent studies demonstrate that the endocannabinoid system influences bone metabolism. With the increasing use of endocannabinoid mimetics, e.g. tetrahydrocannabinol (THC) and cannabidiol (CBD), endocannabinoids' involvement in bone growth and remodeling has become clinically relevant.
Evidence acquisition:
This literature review is based upon a search of Pubmed and Google Scholar databases, as of June 2019, for all English-language publications relating to cannabinoids and bone. We evaluated retrieved articles for relevance, experimental design, data acquisition, statistical analysis, and conclusions.
Evidence synthesis:
Preclinical studies establish a role for endocannabinoids in bone metabolism. These studies yield complex and often contradictory results attributed to differences in the specific experimental model examined. Studies using human cells or subjects are limited.
Conclusions:
In vitro and animal models document that endocannabinoids participate in bone biology. The relevance of these observations to humans is not clear. The increasing chronic use of medical and recreational cannabis underscores the need to better understand the role of endocannabinoids in human bone metabolism. Moreover, it is important to evaluate the role of endocannabinoids as a therapeutic target to prevent and treat disorders associated with bone loss.
... Although our study is not the first to demonstrate that activation of cannabinoid receptors on transformed T cells triggers apoptosis [32,40,43,44], the data presented in this study provide evidence for the first time of a recently synthesized molecule that, by binding CB2R, triggers an apoptotic pathway, involving new insights into the functional roles of cannabinoid receptors. ...
Several studies demonstrated that cannabinoids reduce tumor growth, inhibit angiogenesis, and decrease cancer cell migration. As these molecules are well tolerated, it would be interesting to investigate the potential benefit of newly synthesized compounds, binding cannabinoid receptors (CBRs). In this study, we describe the synthesis and biological effect of 2-oxo-1,8-naphthyridine-3-carboxamide derivative LV50, a new compound with high CB2 receptor (CB2R) affinity. We demonstrated that it decreases viability of Jurkat leukemia cells, evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), but mainly induces a proapoptotic effect. We observed an increase of a hypodiploid peak by propidium iodide staining and changes in nuclear morphology by Hoechst 33258. These data were confirmed by a significant increase of Annexin V staining, cleavage of the nuclear enzyme poly(ADP-ribose)-polymerase (PARP), and caspases activation. In addition, in order to exclude that LV50 non-specifically triggers death of all normal leukocytes, we tested the new compound on normal peripheral blood lymphocytes, excluding the idea of general cytotoxicity. To characterize the involvement of CB2R in the anti-proliferative and proapoptotic effect of LV50, cells were pretreated with a specific CB2R antagonist and the obtained data showed reverse results. Thus, we suggest a link between inhibition of cell survival and proapoptotic activity of the new compound that elicits this effect as selective CB2R agonist.
... This can be due to activation of CB 1 receptors in, for example, C6 glioma cells and primary astrocytes (Blazquez et al., 2000) where it then causes a sustained accumulation of ceramide that activates the JNK/p38 MAPK pathway leading to cell death. Activation of CB 2 receptors, which can also cause ceramide accumulation, has been shown to occur in human Jurkat leukaemia T cells (Herrera et al., 2006), and to lead to mitochondrial-related apoptosis and in human pancreatic tumour cells by a mechanism that involves endoplasmic reticulum stress related genes . However, in colon cancer cells TNF-α has been shown to mediate the de novo synthesis of ceramide, induced by activation of either CB 1 or CB 2 receptors, which then induces apoptosis (Cianchi et al., 2008). ...
Cell fate events are regulated by different endogenous developmental factors such as the cell micro‐environment, external or remote signals and epigenetic factors. Among the many regulatory factors, endocannabinoid‐associated signalling pathways are known to conduct several of these events in the developing nervous system and in the adult brain. Interestingly, endocannabinoids exert modulatory actions in both physiological and pathological conditions. Endocannabinoid signalling can promote cell survival by acting on non‐transformed brain cells (neurons, astrocytes or oligodendrocytes) and can have either a protumoural or antitumoural effect on transformed cells. Moreover, endocannabinoids are able to attenuate the detrimental effects on neurogenesis and neuroinflammation associated with ageing. Thus, the endocannabinoid system emerges as an important regulator of cell fate, controlling cell survival/cell death decisions depending on the cell type and its environment.
Linked Articles
This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc
... Additionally, the stimulation of CB2Rs has been linked to the activation of several intracellular molecules, including the PI3K/AKT pathway (Molina-Holgado et al., 2002;Samson et al., 2003). These pathways have been associated with prosurvival effects and the de novo synthesis of the sphingolipid messenger ceramide (Carracedo, Gironella et al., 2006;Herrera et al., 2006;Sanchez et al., 2001), which has Figure 2.5 CB2R signaling cascade. Δ 9 -tetrahydrocannabinol and cannabinoids also bind to CB2Rs. ...
... Subsequent studies using this cell line and THC, established the intrinsic pathway of apoptosis as being critical (Lombard et al., 2005), but, importantly, that the CB2 receptor did in fact mediate this effect through an increase in p38 MAPK activity (Herrera et al., 2005) and ceramide production (Herrera et al., 2006) and/or through a reduction in the Raf-1/MEK/ERK signaling pathway . With respect to CBD, a meticulous study of CBD-induced apoptosis revealed a dependence on the CB2 receptor for the expression of the NAD(P)H oxidases p22 phox and Nox4, ROS production and caspase activation (McKallip et al., 2006). ...
The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent.
... With respect to SL metabolism, CB2 activation has been shown to induce cell apoptosis through the stimulation of de novo synthesis of Cer in a number of human tumours, including glioma 12,38 , leukemia 39 , and pancreatic cancer 40 . We showed that CB2 activation stimulated Cer synthesis in BC cells thru both conversion of S1P 41 and de novo biosynthesis. ...
The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.
... In fact, blockage of ceramide synthesis confirmed the involvement of ceramide in cannabinoid-induced apoptosis. 31,32 This fact might be related to the non-extenuation of ER-function, thus allowing the ceramide synthesis in the ER. In addition, we detected an early Dwm in treated-MM cells, which occurs even before the cleavage of caspases. ...
... Nevertheless, cannabinoid receptor mediated signaling cannot be viewed mechanistically, as context-dependent decisions are made at signaling crossroads. For example, CB 1 R activation converging onto PI 3 K and ERK activation promotes survival against ceramide-induced apoptosis [355], but chronic CB 1 R and CB 2 R activation itself stimulates the generation of ceramide, which causes sustained ERK activation via RAF proto-oncogene serine/threonine-protein kinase (Raf-1), leading to apoptosis [360,362,363]. CB 1 Rs and CB 2 Rs can modulate intracellular cation levels. ...
Background & objective:
Cannabis is one of the earliest cultivated plants. Cannabis of industrial utility and culinary value is generally termed as hemp. Conversely, cannabis that is bred for medical, spiritual and recreational purposes is called marijuana. The female marijuana plant produces a significant quantity of bio- and psychoactive phytocannabinoids, which regained the spotlight with the discovery of the endocannabinoid system of the animals in the early 90's. Nevertheless, marijuana is surrounded by controversies, debates and misconceptions related to its taxonomic classification, forensic identification, medical potential, legalization and its long-term health consequences.
Method:
In the first part, we provide an in-depth review of the botany and taxonomy of Cannabis. We then overview the biosynthesis of phytocannabinoids within the glandular trichomes with emphasis on the role of peculiar plastids in the production of the secreted material. We also compile the analytical methods used to determine the phytocannabinoid composition of glandular trichomes. In the second part, we revisit the psychobiology and molecular medicine of marijuana.
Results & conclusion:
We summarize our current knowledge on the recreational use of cannabis with respect to the modes of consumption, short-term effects, chronic health consequences and cannabis use disorder. Next, we overview the molecular targets of a dozen major and minor bioactive cannabinoids in the body. This helps us introduce the endocannabinoid system in an unprecedented detail: its up-todate molecular biology, pharmacology, physiology and medical significance, and beyond. In conclusion, we offer an unbiased survey about cannabis to help better weigh its medical value versus the associated risks.
... Indukcja śmierci komórek jest związana ze zmianami morfologicznymi, takimi jak kondensacja chromatyny i fragmentacja DNA oraz zmianami metabolicznymi wynikającymi ze wzrostu aktywności kaspazy 3/7, które są osłabiane przez selektywnych antagonistów receptora CB 1 [35]. Śmierć komórki może być również indukowana aktywacją receptorów CB 2 i TRPV1 [35,44]. ...
Endocannabinoids belong to a group of ester, ether and amide derivatives of fatty acids, which are endogenous ligands of receptors CB1, CB2, TRPV1 and GPR55 that are included in the endocannabinoid system of the animal organism. The best known endocannabinoids are: N-arachidonylethanolamide called anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They occur in all organisms, and their highest level is observed in the brain. In this review the mechanisms of synthesis and degradation of both AEA and 2-AG are shown. Endocannabinoids are synthesized from phospholipids (mainly phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) located in the cell membrane. As a result of arachidonic acid transfer from phosphatidylcholine to phosphatidylethanolamine, N-arachidonoyl phosphatidylethanolamine is formed, which is hydrolyzed to AEA by phospholipase D, C and A2. However, 2-AG is formed during the hydrolysis of phosphatidylinositol catalyzed mainly by DAGL. The primary role of endocannabinoids is the activation of cannabinoid receptors. Both AEA and 2-AG are primarily agonists of the CB1 receptor and to a lower degree CB2 and TRPV1 receptors, but 2-AG has stronger affinity for these receptors. Through activation of receptors, endocannabinoids affect cellular metabolism and participate in the metabolic processes by receptor-independent pathways. Endocannabinoids which are not bound to the receptors are degraded. The main enzymes responsible for the hydrolysis of AEA and 2-AG are FAAH and MAGL, respectively. Apart from hydrolytic degradation, endocannabinoids may also be oxidized by cyclooxygenase-2, lipoxygenases, and cytochrome P450. It has been shown that the metabolites of both endocannabinoids also have biological significance.
The endocannabinoid system is composed by a complex and ubiquitous network of endogenous lipid ligands, enzymes for their synthesis and degradation, and receptors, which can also be stimulated by exogenous compounds, such as those derived from the Cannabis sativa . Cannabis and its bioactive compounds, including cannabinoids and non‐cannabinoids, have been extensively studied in different conditions. Recent data have shown that the endocannabinoid system is responsible for maintaining the homeostasis of various skin functions such as proliferation, differentiation and release of inflammatory mediators. Because of their role in regulating these key processes, cannabinoids have been studied for the treatment of skin cancers and melanoma; their anti‐tumour effects regulate skin cancer progression and are mainly related to the inhibition of tumour growth, proliferation, invasion and angiogenesis, through apoptosis and autophagy induction. This review aims at summarising the current field of research on the potential uses of cannabinoids in the melanoma field.
Introduction: Cannabis use is associated with brain functional changes in regions implicated in prominent neuroscientific theories of addiction. Emerging evidence suggests that cannabidiol (CBD) is neuroprotective and may reverse structural brain changes associated with prolonged heavy cannabis use. In this study, we examine how an ∼10-week exposure of CBD in cannabis users affected resting-state functional connectivity in brain regions functionally altered by cannabis use. Materials and Methods: Eighteen people who use cannabis took part in a ∼10 weeks open-label pragmatic trial of self-administered daily 200 mg CBD in capsules. They were not required to change their cannabis exposure patterns. Participants were assessed at baseline and post-CBD exposure with structural magnetic resonance imaging (MRI) and a functional MRI resting-state task (eyes closed). Seed-based connectivity analyses were run to examine changes in the functional connectivity of a priori regions-the hippocampus and the amygdala. We explored if connectivity changes were associated with cannabinoid exposure (i.e., cumulative cannabis dosage over trial, and plasma CBD concentrations and Δ9-tetrahydrocannabinol (THC) plasma metabolites postexposure), and mental health (i.e., severity of anxiety, depression, and positive psychotic symptom scores), accounting for cigarette exposure in the past month, alcohol standard drinks in the past month and cumulative CBD dose during the trial. Results: Functional connectivity significantly decreased pre-to-post the CBD trial between the anterior hippocampus and precentral gyrus, with a strong effect size (d=1.73). Functional connectivity increased between the amygdala and the lingual gyrus pre-to-post the CBD trial, with a strong effect size (d=1.19). There were no correlations with cannabinoids or mental health symptom scores. Discussion: Prolonged CBD exposure may restore/reduce functional connectivity differences reported in cannabis users. These new findings warrant replication in a larger sample, using robust methodologies-double-blind and placebo-controlled-and in the most vulnerable people who use cannabis, including those with more severe forms of Cannabis Use Disorder and experiencing worse mental health outcomes (e.g., psychosis, depression).
The endocannabinoid system (ECS) comprises two cognate endocannabinoid receptors referred to as CB1R and CB2R. ECS dysregulation is apparent in neurodegenerative/neuro-psychiatric disorders including but not limited to schizophrenia, major depressive disorder and potentially bipolar disorder. The aim of this paper is to review mechanisms whereby both receptors may interact with neuro-immune and neuro-oxidative pathways, which play a pathophysiological role in these disorders. CB1R is located in the presynaptic terminals of GABAergic, glutamatergic, cholinergic, noradrenergic and serotonergic neurons where it regulates the retrograde suppression of neurotransmission. CB1R plays a key role in long-term depression, and, to a lesser extent, long-term potentiation, thereby modulating synaptic transmission and mediating learning and memory. Optimal CB1R activity plays an essential neuroprotective role by providing a defense against the development of glutamate-mediated excitotoxicity, which is achieved, at least in part, by impeding AMPA-mediated increase in intracellular calcium overload and oxidative stress. Moreover, CB1R activity enables optimal neuron-glial communication and the function of the neurovascular unit. CB2R receptors are detected in peripheral immune cells and also in central nervous system regions including the striatum, basal ganglia, frontal cortex, hippocampus, amygdala as well as the ventral tegmental area. CB2R upregulation inhibits the presynaptic release of glutamate in several brain regions. CB2R activation also decreases neuroinflammation partly by mediating the transition from a predominantly neurotoxic “M1” microglial phenotype to a more neuroprotective “M2” phenotype. CB1R and CB2R are thus novel drug targets for the treatment of neuro-immune and neuro-oxidative disorders including schizophrenia and affective disorders.
T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous malignant hematological disorder arising from T-cell progenitors. This study was aimed to evaluate the cytotoxic effect of CP55940 on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat). PBL and Jurkat cells were treated with CP55940 (0-20 µM), and morphological changes in the cell nucleus/ DNA, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by western blotting, pharmacological inhibition and immunofluorescence. CP55940 induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose–response manner with increasing fragmentation of DNA, arrest of cell cycle and damage of ΔΨm. CP55940 increased dichlorofluorescein fluorescence (DCF) intensity, increased DJ-1 Cys¹⁰⁶- sulfonate, a marker of intracellular stress, induced the up-regulation of p53 and phosphorylation of transcription factor c-JUN. It increased the expression of BAX and PUMA, up-regulated mitochondrial proteins PINK1 and Parkin, and activated CASPASE-3. Antioxidant NAC, pifithrin-α, and SP600125 blocked CP55940 deleterious effect on Jurkat cells. However, the potent and highly specific cannabinoid CB1 and CB2 receptor inverse agonist SR141716 and SR144528 were unable to blunt CP55940-induced apoptosis in Jurkat cells. Conclusively CP55940 provokes cell death in Jurkat through CBR-independent mechanism. Interestingly, CP55940 was also cytotoxic to ex vivo T-ALL cells from chemotherapy-resistant pediatric patients. In conclusion, CP55940 selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway. Our findings support the use of cannabinoids as a potential treatment for T-ALL cells.
: Thanks to the success of modern antiretroviral therapy (ART), people living with HIV (PLWH) have life expectancies which approach that of persons in the general population. However, despite the ability of ART to suppress viral replication, PLWH have high levels of chronic systemic inflammation which drives the development of comorbidities such as cardiovascular disease, diabetes and non-AIDS associated malignancies. Historically, cannabis has played an important role in alleviating many symptoms experienced by persons with advanced HIV infection in the pre-ART era and continues to be used by many PLWH in the ART era, though for different reasons. Δ-tetrahydrocannabinol (Δ-THC) and cannabidiol (CBD) are the phytocannabinoids which have received most attention for their medicinal properties. Due to their ability to suppress lymphocyte proliferation and inflammatory cytokine production, there is interest in examining their therapeutic potential as immunomodulators. CB2 receptor activation has been shown in vitro to reduce CD4 T-cell infection by CXCR4-tropic HIV and to reduce HIV replication. Studies involving SIV-infected macaques have shown that Δ-THC can reduce morbidity and mortality and has favourable effects on the gut mucosal immunity. Furthermore, ΔTHC administration was associated with reduced lymph node fibrosis and diminished levels of SIV proviral DNA in spleens of rhesus macaques compared with placebo-treated macaques. In humans, cannabis use does not induce a reduction in peripheral CD4 T-cell count or loss of HIV virological control in cross-sectional studies. Rather, cannabis use in ART-treated PLWH was associated with decreased levels of T-cell activation, inflammatory monocytes and pro-inflammatory cytokines secretion, all of which are related to HIV disease progression and co-morbidities. Randomized clinical trials should provide further insights into the ability of cannabis and cannabinoid-based medicines to attenuate HIV-associated inflammation. In turn, these findings may provide a novel means to reduce morbidity and mortality in PLWH as adjunctive agents to ART.
The endocannabinoid system (ECS) consists of the endocannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade endocannabinoids. The whole EC system plays an important role in the proper functioning of the central and autonomic nervous system. ECS is involved in the regulation of the body energy and in the functioning of the endocrine system. It can affect on the regulation of emotional states, motoric movement, operations of the endocrine, immune and digestive system. Many of the effects of cannabinoids are mediated by G coupled –protein receptors: CB1, CB2 and GPR55 but also of transient receptor potential channels (TRPs) which not only induce the sensation of pain but also support inflammation via secretion of pro-inflammatory neuropeptides. In this review work we briefly summarize the role and action of cannabinoid receptors CB1 and CB2, protein-coupled receptor 55 (GPR55) and transient receptor potential vanilloid 1 (TRPV1).
There has been a substantial increase, worldwide, in the incidence of thyroid cancer over the past few decades. The diagnosis of thyroid cancer poses a clinical challenge to medical practitioners. Fine needle aspiration cytology (FNAC) is a current standard method to diagnose thyroid cancer. FNAC has a false-positive rate of 1% - 5% and a false-negative rate of 0% - 7.7%, whilst 20% - 30% of cases remain non-diagnostic. Grey-scale ultrasound is a useful imaging technique for the assessment of the thyroid gland, because it can characterize various features of thyroid nodules such as the number, site, size, shape, echogenicity, and internal architecture. However, grey-scale ultrasound has limited value in differentiating benign and malignant thyroid nodules because of its varied sensitivity (52% - 97%) and specificity (26% - 83%). The varied sensitivity and specificity of grey-scale ultrasound might be due to the qualitative analysis and subjective interpretation of the characteristics of thyroid nodules and renders it thus vulnerable to inter- and intra-observer variations. Nevertheless, certain features of thyroid nodules can be quantified, for example tissue stiffness and the vascularity index. Shear wave elastography is a novel ultrasound technique that can quantify tissue stiffness by tracking the propagation of shear waves through thyroid nodules. In general, malignant thyroid nodules tend to be stiffer than benign nodules. Depending upon the differences in stiffness values, thyroid nodules can be differentiated as benign or malignant. The first study of this thesis evaluated the feasibility of using shear wave elastography in predicting thyroid malignancy, and determined if any (and if so, which ones) of the shear wave elastography indices (Emaximum, Emean, and Eminimum) are potential predictors of thyroid malignancy. The study further evaluated the diagnostic accuracy in differentiating benign and malignant thyroid nodules when grey-scale ultrasound was combined with shear wave elastography. Vascularity is a dynamic feature usually detected on Doppler ultrasound. Central vascularity and hypervascularity are usually associated with malignant thyroid nodules. However, not all previous studies appreciate the usefulness of vascularity in predicting thyroid malignancy. Controversial results might be due to the fact that previous studies evaluated vascularity by visual assessment methods that involve subjective interpretation and thus result in high inter- and intra-observer variations. Moreover, in previous studies, the methods used to delineate a border between peripheral and central regions of a thyroid nodule were not standardized. In the second study in this thesis, we have thus developed a computer algorithm that can perform regional segmentation of thyroid nodules by using an 'offsetting' method, and quantify the overall vascularity as well as the vascularity in peripheral and central regions of thyroid nodules. Based on the differences in the vascularity indices, thyroid nodules can be differentiated into benign and malignant types. The study further evaluated the potential advantage of combining the vascularity index with grey-scale ultrasound to enhance the diagnostic accuracy of thyroid malignancy. In the first and second study, a total of 111 patients with solitary thyroid nodules were included. Each thyroid nodule was assessed with grey-scale ultrasound, shear wave elastography, and colour Doppler ultrasound. The diagnosis of the thyroid nodules was confirmed by FNAC and/or histological examination. Grey-scale ultrasound features including microcalcification (malignant: 77.8% versus benign: 7.1%), hypoechoic (92.6% versus 33.3%), irregular margins (55.6% versus 16.7%), and a height-to-width ratio > 1 (59.3% versus 13.1%) were found to be more frequently associated with malignant thyroid nodules than with benign thyroid nodules. The differences were statistically significant (all P < 0.05). Regarding shear wave elastography, the results suggested that Emaximum ≥ 67.3 kPa and Emean ≥ 23.1 kPa are independent predictors of thyroid malignancy. Emaximum was found to be the best adjunct to grey-scale ultrasound. The combination of Emaximum or Emean with grey-scale ultrasound enhanced the diagnostic accuracy of grey-scale ultrasound from 58.5% to 80.2% and 78.4%, respectively (P < 0.05). The results of the vascularity index (VI) quantification showed that a 22% offset was optimal for regional subdivision of thyroid nodules. At the optimum offset, the mean VI of peripheral, central, and overall regions of malignant nodules were significantly higher than those of benign nodules (26.5 ± 16.2%, 21.7 ± 19.6%, 23.8 ± 4.6% versus 18.2 ± 16.7%, 11.9 ± 15.1%, and 16.6 ± 1.8%, respectively, P < 0.05). The optimum cut-off points of peripheral, central, and overall VI were 19.7%, 9.1%, and 20.2%, respectively. When compared to grey-scale ultrasound alone, a combination of VI assessment and grey-scale ultrasound evaluation of thyroid nodules increased the overall diagnostic accuracy from 58.6% to 79.3% (P < 0.05). Studies 1 and 2 have clinical significance in establishing methods for accurate diagnosis of thyroid cancer. The results of Study 1 suggest that shear wave elastography has clinical importance in differentiating benign and malignant thyroid nodules. The combination of grey-scale ultrasound with Emaximum or Emean significantly improved the diagnostic accuracy in predicting thyroid cancer. Study 2 has devised a new method to perform regional segmentation of thyroid nodules and to quantify vascularity in each segment. This approach is objective and standardized to quantify thyroid vascularity and helps in differentiating benign and malignant thyroid nodules.
Colour Doppler ultrasound (CDU) and power Doppler ultrasound (PDU) are widely used for detecting the vasculature of tissues or organs. In the thyroid gland, CDU and PDU have been used for the differential diagnosis of, amongst others, Hashimoto's disease, Graves' disease, and Reidel's thyroiditis. Doppler ultrasound has also been used in monitoring treatment responses during therapy of thyroid disorders. However, the detection of blood flow in minute blood vessels or vessels with low blood flow is challenging due to technical limitations of previously developed Doppler ultrasound modalities. Most recently, a new ultrasound technology, namely AngioPLUS (Planewave UltraSensitive™ imaging), provides superb sensitivity in the detection of tissue vascularity. The beauty of this technique is that all colour pixels of the tissue can be reconstructed in a single image. AngioPLUS provides high resolution and 3D wall filtering that allow efficient discrimination between blood flow and other soft tissues by analysing space, time, and amplitude information. Study 3 aimed to evaluate the feasibility of using AngioPLUS imaging in assessing thyroid vascularity when combined with CDU or PDU. The study further evaluated whether the addition of AngioPLUS to CDU or PDU enhances the sensitivity in detecting vasculature of thyroid parenchyma. It also investigated whether there is any asymmetry of vascularity between the right and left thyroid lobes. A total of 45 healthy volunteers underwent grey-scale ultrasound, CDU, CDU+AngioPLUS, PDU, and PDU+AngioPLUS evaluations of both lobes of the thyroid gland. Thyroid vascularity was evaluated using our in-house computer algorithm. The results showed that the combination of CDU+AngioPLUS (14.7 ± 9.4%) and the combination of PDU+AngioPLUS (13.4 ± 9%) had significantly higher thyroid VI than CDU (8.8 ± 7.3%) and PDU (4.7 ± 5.4%) alone (all P < 0.05). No asymmetry was found between the VI of the right and left thyroid lobes (P > 0.05). Study 3 highlights the differences in sensitivity of detecting thyroid vasculature assessed by different Doppler ultrasound modalities. The results suggest that AngioPLUS enhances the detection of vascularity when added to PDU or CDU. The clinical significance of the study lies in the detection of small blood vessels and vessels with low blood flow that may help disease diagnosis and treatment monitoring. Study 4 highlights the scope of chemotherapeutic agents in treating papillary thyroid cancer. Current strategies to treat papillary thyroid cancer are largely based on surgery, where recurrence rate is high (up to 33%). Drug development is a costly and time-consuming process. Moreover, the identification of novel therapeutic targets is challenging. Study 4 introduces the concept of 'repurposing of drugs' that evaluates the therapeutic potential of already approved drugs beyond the scope of their primary clinical usage. Cannabinoids are derivatives of the marijuana plant. They have been used for recreation and to relieve pain. Two cannabinoid receptors (CB1 and CB2) are known to be distributed over different body organs and systems in humans. Cannabinoids receptor expression has been noted in many cancers including breast cancer, prostate cancer, hepatic cancer, lung cancer, and colorectal cancer. Anti-cancer actions, including antiproliferation, antimigration, antiangiogenesis, and apoptosis, have been validated in in vitro experiments in various cancer cell lines. In a recent study, immunohistochemistry analysis of surgical specimens of 87 thyroid nodules demonstrated that CB1 and CB2 receptor expression was more significantly associated with papillary thyroid cancer than with benign thyroid nodules. It was further found that CB2 expression was significantly higher than CB1 receptor expression. In Study 4, the therapeutic potential of CB2 receptor agonist (JWH-133) in treating papillary thyroid cancer was evaluated. A normal thyroid follicular cell line (N-thy-ori-3) was used as the control, and BCPAP was the papillary thyroid cancer cell line used in this study. Both cell lines were treated with JWH-133 at 0, 5, 10, 15, 20, 25, and 30-µM concentrations for 24, 48, and 72 hours. Cellular metabolic activity and cell viability were evaluated using an MTT assay. The results demonstrated that 25 µM was the lethal dose concentration for BCPAP cells at which cell viability was reduced to 50% after the optimal 48 hours of incubation. The results also suggested that BCPAP cells were more sensitive to the JWH-133 as compared with N-thy-ori-3. The cytotoxic effect mediated by JWH-133 was not significantly inhibited by CB2 receptor antagonist SR144528 in both cell lines. The results of the study demonstrated that JWH-133 has a potent cytotoxic effect, more pronounced in a papillary thyroid cancer cell line (BCPAP) than in a normal follicular thyroid cell line (N-thy-ori-3). Study 4 suggests that JWH-133 induces cell death in papillary thyroid cancer cells, whilst the survival of normal thyroid follicular cells can be maintained at an acceptable level.
Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the active component of Cannabis sativa preparations, such as hashish and marijuana, signals through cell surface receptors. Two types of cannabinoid receptors have been cloned and characterized. CB1 receptors are ubiquitously expressed in the neurons of the brain. They are presynaptically expressed in both inhibitory interneurons and excitatory neurons. CB2 receptors are expressed in microglial cells, astrocytes, and several neuron subpopulations. They are expressed in both the pre- and postsynaptic terminals. The present chapter discusses the current understanding of the cannabinoid receptors and their signaling cascades.
Cannabinoid receptors have been shown to interact with other receptors, including Tumor Necrosis Factor Receptor Superfamily (TNFRS) members, to induce cancer cell death. When cannabinoids and death-inducing ligands (including TRAIL) are administered together, they have been shown to synergize and demonstrate enhanced antitumor activity in vitro. Certain cannabinoid ligands have been shown to sensitize cancer cells and synergistically interact with members of the TNFRS, thus suggesting that the combination of cannabinoids with death receptor (DR) ligands induces additive or synergistic tumor cell death. This review summarizes recent findings on the interaction of the cannabinoid and DR systems and suggests possible clinical co-application of cannabinoids and DR ligands in the treatment of various malignancies. This article is protected by copyright. All rights reserved.
Endocannabinoids are bioactive lipids that modulate various physiological processes through G-protein-coupled receptors (CB1 and CB2) and other putative targets. By sharing the activation of the same receptors, some phytocannabinoids and a multitude of synthetic cannabinoids mimic the effects of endocannabinoids. In recent years, a growing interest has been dedicated to the study of cannabinoids properties for their analgesic, antioxidant, anti-inflammatory and neuroprotective effects. In addition to these well-recognized effects, various studies suggest that cannabinoids may affect cell survival, cell proliferation or cell death. These observations indicate that cannabinoids may play an important role in the regulation of cellular homeostasis and, thus, may contribute to tissue remodelling and cancer treatment. For a long time, the study of cannabinoid receptor signalling has been focused on the classical adenylyl cyclase/cyclic AMP/protein kinase A (PKA) pathway. However, this pathway does not totally explain the wide array of biological responses to cannabinoids. In addition, the diversity of receptors and signalling pathways that endocannabinoids modulate offers an interesting opportunity for the development of specific molecules to disturb selectively the endogenous system. Moreover, emerging evidences suggest that cannabinoids ability to limit cell proliferation and to induce tumour-selective cell death may offer a novel strategy in cancer treatment. This review describes the main properties of cannabinoids in cell death and attempts to clarify the different pathways triggered by these compounds that may help to understand the complexity of respective molecular mechanisms and explore the potential clinical benefit of cannabinoids use in cancer therapies.
Cannabis is the most popular illicit drug in the Western world, and has a long history of medical use. Its active ingredients, known as cannabinoids, can elicit various biological activities through activation of certain G-protein coupled receptors, denoted by the name of cannabinoid receptors. By far, two cannabinoid receptors have been identified, namely the cannabinoid receptor 1 (CB1) and 2 (CB2). The CB1 receptors are primarily found in the central nervous system (CNS) while the CB2 receptors largely present in the periphery. Besides the plant-derived cannabinoids (phytocannabinoids), a group of endogenous lipid-derived molecules can similarly activate the cannabinoid receptors; therefore they are referred to as endocannabinoids. Activation and blockade of cannabinoid receptors can lead to a variety of responses relevant to pharmacotherapy. This chapter provides a synopsis of phytocannabinoids, and an overview of cannabinoid receptors with respects to: what they are; where they are; and what they do.
Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528. Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.
We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).
Anandamide was the first brain metabolite shown to act as a ligand of “central” CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 μM and 83–92% maximal inhibition at 5–10 μM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 μM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1–0.5 μM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.
Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands
for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry
and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a
radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent
inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids.
These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.
Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.
The major active ingredient of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and delta 9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of delta 9-THC, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the nonpsychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.
The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.
Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide
increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted
from the inhibition of adenylyl cyclase and cyclic adenosine 3,5-monophosphate-dependent protein kinase. One of the proteins
phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in
neurons. Focal adhesion kinaseis a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton.
Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.
Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin,
but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective
in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation
of sphingolipids. Selection of lysenin-resistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient
mutants, both of which showed less lysenin binding capability than wild-type cells. One mutant strain was severely defective
in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable ofde novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection
of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein
in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein
antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface
sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient
mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.
Delta9-Tetrahydrocannabinol, the main active component of marijuana, induces apoptosis of transformed neural cells in culture. Here, we show that intratumoral administration of Delta9-tetrahydrocannabinol and the synthetic cannabinoid agonist WIN-55,212-2 induced a considerable regression of malignant gliomas in Wistar rats and in mice deficient in recombination activating gene 2. Cannabinoid treatment did not produce any substantial neurotoxic effect in the conditions used. Experiments with two subclones of C6 glioma cells in culture showed that cannabinoids signal apoptosis by a pathway involving cannabinoid receptors, sustained ceramide accumulation and Raf1/extracellular signal-regulated kinase activation. These results may provide the basis for a new therapeutic approach for the treatment of malignant gliomas.
Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the
death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide
preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The
caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine,
whereas overexpression of Bcl-xL had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable
C2-ceramide, induced cytochromec release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and,
surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely
abolished by Bcl-xL overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced
cell death in type II cells.
Cannabinoids exert most of their effects in the central nervous system through the CB(1) cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin and wortmannin blocked the CB(1) receptor-evoked activation of PKB, pointing to the sequential involvement of a G(i)/G(o) protein and phosphoinositide 3'-kinase. The functionality of the cannabinoid-induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB(1) receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB(2) receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB(1) receptor.
B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.
Cannabinoids exert most of their effects through the CB(1) receptor. This G protein-coupled receptor signals inhibition of adenylyl cyclase, modulation of ion channels, and stimulation of mitogen- and stress-activated protein kinases. In this article, we report that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces sphingomyelin hydrolysis in primary astrocytes but not in other cells expressing the CB(1) receptor, such as primary neurons, U373 MG astrocytoma cells, and Chinese hamster ovary cells transfected with the CB(1) receptor cDNA. THC-evoked sphingomyelin breakdown in astrocytes was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoid HU-210 and was prevented by the selective CB(1) antagonist SR141716. By contrast, the effect of THC was not blocked by pertussis toxin, pointing to a lack of involvement of G(i/o) proteins. A role for the adaptor protein FAN in CB(1) receptor-coupled sphingomyelin breakdown is supported by two observations: 1) coimmunoprecipitation experiments show that the binding of FAN to the CB(1) receptor is enhanced by THC and prevented by SR141716; 2) cells expressing a dominant-negative form of FAN are refractory to THC-induced sphingomyelin breakdown. This is the first report showing that a G-protein-coupled receptor induces sphingomyelin hydrolysis through FAN and that the CB(1) cannabinoid receptor may signal independently of G(i/o) proteins.
The development of new therapeutic strategies is essential for the management of gliomas, one of the most malignant forms of cancer. We have shown previously that the growth of the rat glioma C6 cell line is inhibited by psychoactive cannabinoids (I. Galve-Roperh et al., Nat. Med., 6: 313-319, 2000). These compounds act on the brain and some other organs through the widely expressed CB(1) receptor. By contrast, the other cannabinoid receptor subtype, the CB(2) receptor, shows a much more restricted distribution and is absent from normal brain. Here we show that local administration of the selective CB(2) agonist JWH-133 at 50 microg/day to Rag-2(-/-) mice induced a considerable regression of malignant tumors generated by inoculation of C6 glioma cells. The selective involvement of the CB(2) receptor in this action was evidenced by: (a) the prevention by the CB(2) antagonist SR144528 but not the CB(1) antagonist SR141716; (b) the down-regulation of the CB(2) receptor but not the CB(1) receptor in the tumors; and (c) the absence of typical CB(1)-mediated psychotropic side effects. Cannabinoid receptor expression was subsequently examined in biopsies from human astrocytomas. A full 70% (26 of 37) of the human astrocytomas analyzed expressed significant levels of cannabinoid receptors. Of interest, the extent of CB(2) receptor expression was directly related with tumor malignancy. In addition, the growth of grade IV human astrocytoma cells in Rag-2(-/-) mice was completely blocked by JWH-133 administration at 50 microg/day. Experiments carried out with C6 glioma cells in culture evidenced the internalization of the CB(2) but not the CB(1) receptor upon JWH-133 challenge and showed that selective activation of the CB(2) receptor signaled apoptosis via enhanced ceramide synthesis de novo. These results support a therapeutic approach for the treatment of malignant gliomas devoid of psychotropic side effects.
Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.
Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.
In the current study, we examined whether ligation of CB2 receptors would lead to induction of apoptosis in tumors of immune origin and whether CB2 agonist could be used to treat such cancers. Exposure of murine tumors EL-4, LSA, and P815 to delta-9-tetrahydrocannabinol (THC) in vitro led to a significant reduction in cell viability and an increase in apoptosis. Exposure of EL-4 tumor cells to the synthetic cannabinoid HU-210 and the endogenous cannabinoid anandamide led to significant induction of apoptosis, whereas exposure to WIN55212 was not effective. Treatment of EL-4 tumor-bearing mice with THC in vivo led to a significant reduction in tumor load, increase in tumor-cell apoptosis, and increase in survival of tumor-bearing mice. Examination of a number of human leukemia and lymphoma cell lines, including Jurkat, Molt-4, and Sup-T1, revealed that they expressed CB2 receptors but not CB1. These human tumor cells were also susceptible to apoptosis induced by THC, HU-210, anandamide, and the CB2-selective agonist JWH-015. This effect was mediated at least in part through the CB2 receptors because pretreatment with the CB2 antagonist SR144528 partially reversed the THC-induced apoptosis. Culture of primary acute lymphoblastic leukemia cells with THC in vitro reduced cell viability and induced apoptosis. Together, the current data demonstrate that CB2 cannabinoid receptors expressed on malignancies of the immune system may serve as potential targets for the induction of apoptosis. Also, because CB2 agonists lack psychotropic effects, they may serve as novel anticancer agents to selectively target and kill tumors of immune origin.
Hereditary sensory neuropathy type 1 (HSN1) is a dominantly inherited degenerative disorder of the peripheral nerves. HSN1 is clinically and genetically heterogeneous. One form arises from mutations in the gene SPTLC1 encoding long-chain base 1 (LCB1), one of two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step of sphingolipid synthesis. We have examined the effects of the mutations C133Y and C133W, which we have identified in two HSN1 families, on the function of SPT. Although in HSN1 lymphoblasts, the C133Y and C133W mutations do not alter the steady-state levels of LCB1 and LCB2 subunits, they result in reduced SPT activity and sphingolipid synthesis. Moreover, in a mutant Chinese hamster ovary (CHO) cell strain with defective SPT activity due to a lack of the LCB1 subunit, these mutations impair the ability of the LCB1 subunit to complement the SPT deficiency. Furthermore, the overproduction of either the LCB1C133Y or LCB1C133W subunit inhibits SPT activity in CHO cells despite the presence of wild-type LCB1. In addition, we demonstrate that in CHO cells the mutant LCB1 proteins, similar to the normal LCB1, can interact with the wild-type LCB2 subunit. These results indicate that the HSN1-associated mutations in LCB1 confer dominant negative effects on the SPT enzyme.
Nonmelanoma skin cancer is one of the most common malignancies in humans. Different therapeutic strategies for the treatment of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the wide tissue distribution of the two subtypes of cannabinoid receptors (CB(1) and CB(2)), we studied the potential utility of these compounds in anti-skin tumor therapy. Here we show that the CB(1) and the CB(2) receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic death of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CB(1)/CB(2) agonist WIN-55,212-2 or the selective CB(2) agonist JWH-133 induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by altered blood vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth factor, and angiopoietin 2). Abrogation of EGF-R function was also observed in cannabinoid-treated tumors. These results support a new therapeutic approach for the treatment of skin tumors.
Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression in rodents (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell migration and survival as well as the decrease of the expression of proangiogenic factors (vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of cannabinoid-based antitumoral therapies.
Stimulation of cannabinoid CB1 receptors by 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA) inhibits the growth of a rat thyroid cancer cell-derived tumor in athymic mice by inhibiting the activity of the oncogene product p21ras. Here we report that Met-F-AEA also blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat thyroid carcinoma cells. Met-F-AEA significantly inhibited, in tumors as well as transformed cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to be up-regulated by p21ras, as well as of one of its receptors, flt-1/VEGFR-1. The levels of the cyclin-dependent kinase inhibitor p27(kip1), which is down-regulated by p21ras, were instead increased by Met-F-AEA. All these effects were antagonized by the selective CB1 receptor antagonist SR141716A. Met-F-AEA inhibited in vitro the growth of a metastasis-derived thyroid cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1 receptor stimulation interferes not only with angiogenesis but also with metastatic processes was tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL) in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met-F-AEA reduced significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo by inhibiting at once tumor growth, angiogenesis, and metastasis.
The endocannabinoid system is still poorly understood. Recently, the basic elements that constitute it, i.e., membrane receptors, endogenous ligands, and mechanisms for termination of the signaling process, have been partially characterized. There is a considerable lack of information, however, concerning the distribution, concentration, and function of those components in the human body, particularly during pathological events. We have studied the status of some of the components of the endocannabinoid system, fatty acid amide hydrolase and cannabinoid CB1 and CB2 receptors, in postmortem brains from patients with Alzheimer's disease. Using specific polyclonal antibodies, we have performed immunohistochemical analysis in hippocampus and entorhinal cortex sections from brains of Alzheimer's disease patients. Our results show that both fatty acid amide hydrolase and cannabinoid CB2 receptors are abundantly and selectively expressed in neuritic plaque-associated astrocytes and microglia, respectively, whereas the expression of CB1 receptors remains unchanged. In addition, the hydrolase activity seems to be elevated in the plaques and surrounding areas. Thus, some elements of the endocannabinoid system may be postulated as possible modulators of the inflammatory response associated with this neurodegenerative process and as possible targets for new therapeutic approaches.
Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Delta9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.